The serpin ␣ 2 -antiplasmin (SERPINF2) is the principal inhibitor of plasmin and inhibits fibrinolysis. Accordingly, ␣ 2 -antiplasmin deficiency in humans results in uncontrolled fibrinolysis and a bleeding disorder. ␣ 2 -antiplasmin is an unusual serpin, in that it contains extensive Nand C-terminal sequences flanking the serpin domain. The N-terminal sequence is crosslinked to fibrin by factor XIIIa, whereas the C-terminal region mediates the initial interaction with plasmin. To understand how this may happen, we have determined the 2.65Å X-ray crystal structure of an N-terminal truncated murine ␣ 2 -antiplasmin. The structure reveals that part of the C-terminal sequence is tightly associated with the body of the IntroductionVertebrate vascular integrity is protected by a sophisticated hemostatic mechanism that, when activated by trauma, leads to the formation of a fibrin-rich clot. Simultaneously the fibrinolytic system is activated to begin the process of remodelling and removing the clot during tissue repair. 1 Fibrinolysis is initiated by trace quantities of tissue plasminogen activator (tPA) derived from endothelial cells. In the presence of fibrin, tPA cleaves the protease plasminogen (which comprises an apple domain, 5 kringle domains [K1-K5], and a C-terminal serine protease domain 2 ) between the fifth kringle domain and the protease domain to form plasmin, which mediates clot lysis. 3 All 6 domains of plasmin remain associated via a disulphide bond after cleavage. 4 The physiologic inhibitor of plasmin (k a 3.8 ϫ 10 7 M Ϫ1 s Ϫ1 ) 5 is ␣ 2 -antiplasmin and patients deficient in this serpin suffer a variable, but often severe, bleeding disorder. 6,7 By contrast, mice with a targeted deletion of ␣ 2 -antiplasmin have a normal hemostatic response to minor trauma, presumably because the deficient animals plasma contained significant residual plasmin inhibitory activity. 8 However, when challenged with artificially induced pulmonary emboli, the deficient mice have a greater survival rate than the wild type (41.7% mortality vs 68.8%) consistent with up-regulation of the fibrinolytic system. 9 These data suggest that therapeutic intervention in the plasmin/␣ 2 -antiplasmin interaction may be of benefit to patients with thrombotic disorders.␣ 2 -antiplasmin contains extensive N-and C-terminal sequences that flank the serpin domain ( Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). The N-terminal sequence is crosslinked to fibrin by factor XIIIa. 10 The 55 amino acid C-terminal sequence binds to the K1 and K4 domains of plasmin most strongly (K2 and K5 with lower affinity) 8,11 and enhances the rate of interaction between plasmin and ␣ 2 -antiplasmin by 30-to 60-fold. 5 It is suggested that the C-terminus acts as a template for the interaction with plasmin, bringing its active site into apposition with the serpin reactive site. 8 To understand the role of ␣ 2 -antiplasmin in regulating fibrinolysis and the function of the C-terminus we report th...

␣ 2 -Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N-and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of ␣ 2 -antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of ␣ 2 -antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between ␣ 2 -antiplasmin and plasmin. When the C terminus of ␣ 2 -antiplasmin was removed, the binding affinity for active siteblocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.

Legionella pneumophila is an opportunistic pathogen that replicates within alveolar macrophages resulting in the onset of severe atypical pneumonia. Previously we have identified Lpg1905, a eukaryotic-type ecto-NTPDase (nucleoside triphosphate diphosphohydrolase) from L. pneumophila that was required for optimal intracellular replication and virulence in a mouse lung infection model. In the present study, we characterized the activity of a second eukaryotic-type NTPDase, Lpg0971, from L. pneumophila. We observed that recombinant Lpg0971 hydrolysed only ATP and exhibited divalent cation preference for manganese (II) ions. Similar to lpg1905, an lpg0971 mutant carrying the plasmid pMIP was attenuated in a mouse lung infection model and impaired for replication in human macrophages and amoebae. Increased trafficking of the LCV (Legionella-containing vacuole) to a LAMP-1 (lysosome-associated membrane protein-1)-positive compartment was observed for both the lpg1905 and lpg0971 mutants carrying pMIP. Complementation with either lpg1905 or lpg0971 restored intracellular replication, suggesting that a minimum level of ATPase activity was required for this function. A double lpg1905/0971 mutant was not more impaired for intracellular replication than the single mutants and complementation of the double mutant with lpg0971, but not lpg1905, restored intracellular replication. This suggested that although the NTPDases have overlapping activities they have distinct functions. Unlike many eukaryotic-type proteins from L. pneumophila, neither Lpg1905 nor Lpg0971 were translocated into the host cell by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IV secretion system. Overall our data suggest that the ability of L. pneumophila to replicate in eukaryotic cells relies in part on the ability of the pathogen to hydrolyse ATP within an intracellular compartment.